Baumstark J S
Department of Obstetrics and Gynecology, Creighton University, School of Medicine, Omaha, NE 68131.
J Biochem Biophys Methods. 1987 Apr;14(2):59-70. doi: 10.1016/0165-022x(87)90041-8.
A rapid method for the separation of transferrin from swine or human serum is described. Serum (human or swine) is brought to 50% of saturation with ammonium sulfate for removal of immunoglobulins, the resulting precipitate discarded and the supernatant brought to 70% of saturation. The resulting precipitate was dissolved in and dialyzed against 1.54 mM sodium azide (I = 0.00154). Chromatography of the low ionic strength ammonium sulfate fractions (= 20 ml of swine or human serum, 70% of saturation) on columns of Bio-Gel A-1.5 m-Reactive Blue 2, equilibrated with 1.54 mM sodium azide, resulted in two peaks, a breakthrough peak and pure transferrin which was eluted with a linear gradient with 0.5 M potassium phosphate buffer, pH 7.1, as limit buffer. Yields varied between 53 and 55% from whole serum and 70-76% from the ammonium sulfate fractions. Transferrins from both species were found to be homogeneous when subjected to immunoelectrophoresis (anti whole serum antibody) and anionic and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. Hemopexin, a frequently found contaminant in transferrin preparations, is tightly bound by the gel-dye complex under the experimental conditions. Swine serum transferrin possesses many physicochemical properties practically identical to the human protein. Although small differences in physicochemical properties were apparent the extinction coefficients, molecular weights, electrophoretic mobilities, absorbance maxima of the diferric proteins (470 nm), isoelectric points and the absorbance ratios (465 nm/410 nm) of the diferric proteins were practically identical. Both swine and human transferrin produced a reaction of identity (complete coalescence) when reacted with antibody to either transferrin.
本文描述了一种从猪或人血清中分离转铁蛋白的快速方法。血清（人或猪）用硫酸铵使其饱和度达到50%以去除免疫球蛋白，将所得沉淀弃去，上清液再达到70%饱和度。所得沉淀溶解于1.54 mM叠氮化钠（离子强度I = 0.00154）中并进行透析。将低离子强度硫酸铵级分（相当于20 ml猪或人血清，70%饱和度）在Bio - Gel A - 1.5m - 活性蓝2柱上进行色谱分析，该柱用1.54 mM叠氮化钠平衡，得到两个峰，一个穿透峰和纯转铁蛋白，纯转铁蛋白用0.5 M磷酸钾缓冲液（pH 7.1）作为极限缓冲液进行线性梯度洗脱。全血清的产率在53%至55%之间，硫酸铵级分的产率在70%至76%之间。当进行免疫电泳（抗全血清抗体）以及阴离子和十二烷基硫酸钠聚丙烯酰胺圆盘凝胶电泳时，发现两种来源的转铁蛋白都是均一的。在实验条件下，转铁蛋白制剂中常见的污染物血红素结合蛋白与凝胶 - 染料复合物紧密结合。猪血清转铁蛋白具有许多与人类蛋白质几乎相同的物理化学性质。尽管在物理化学性质上存在一些细微差异，但二价铁蛋白的消光系数、分子量、电泳迁移率、最大吸收波长（470 nm）、等电点以及二价铁蛋白的吸收比（465 nm/410 nm）实际上是相同的。当与抗转铁蛋白抗体反应时，猪和人转铁蛋白都产生同一反应（完全融合）。
